Cerebrovascular disease is the third leading cause of morbidity and mortality in the United States. As the population ages, it will be even more pressing to develop effective treatment options that can not only increase survival, but decrease disability. In a rat model of stroke (the middle cerebral artery occlusion or MCAO) we found that intravenously (iv) administering the mononuclear fraction from human umbilical cord blood (HUCB) enhances motor functions. In this proposal we will explore how the administration of this population of cells may induce recovery and decrease anatomical damage induced by MCAO through direct neuroprotective and anti-inflammatory mechanisms. In Aim 1we will characterize how iv HUCB cells modify the underlying pathology and inflammation of the MCAO when administered 24 hours to 7 days after the MCAO using markers of inflammation, neuronal death, neurosurvival, and apoptosis. In Aim 2, we will identify the specific subpopulation of the HUCB cells that is instrumental in inducing behavioral and anatomical recovery. This will be accomplished using fluorescent activated cell sorting (FACS) to enrich T cell, B Cell, monocyte and "stem" cell fractions for transplantation into a stroked rat. Endpoint measures in these studies will include performance on a battery of motor tests and infarct volume. Aim 3 proposes a series of in vitro studies to examine whether the HUCB cells modify the inflammatory response to the MCAO through direct interactions with neural cell popultions. Cell culture assays using neuronal, astrocytic, oligodendrocytes or microglia will be conducted to identify direct interactions and the molecular signals that mediate survival, cytokine expression and NF-kappaB binding activity in response to hypoxia/reoxygenation. In Aim 4 we will use microarray technology to identify neural repair/survival genes that are up-regulated by HUCB treatment of neuronal cultures. All microarray experiments will be verified with western blots and immunohistochemistry. The studies proposed here will increase our understanding of the neuroprotective and anti-inflammatory mechanisms underlying the recovery from stroke induced by HUCB cell transplantation and may also identify other potential targets in stroke for development of treatment options. The ultimate goal of this research program is to develop an innovative HUCB cell based therapy into a viable clinical option for transplantation in neurodegenerative disease and brain injury.